Salt and polymorphs of a kinesin inhibitor compound

ABSTRACT

The present invention relates to salts, polymorphs and hydrates of the kinesin inhibitor compound N-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamide, processes to prepare such salts, polymorphs and hydrates and a liquid formulation comprising at least one of these salts, polymorphs and hydrates.

The present invention relates to salts and polymorphs of the kinesininhibitor compoundN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamide.

Kinesins are motor proteins that hydrolyze adenosine triphosphate asthey travel along microtubules and generate mechanical force. Theseproteins are characterized by containing a motor domain having about 350amino acid residues. The crystal structures of several kinesin motordomains have been resolved.

Currently, about one hundred kinesin-related proteins (KRP) have beenidentified. Kinesins are involved in a variety of cell biologicalprocesses including transport of organelles and vesicles, andmaintenance of the endoplasmic reticulum. Several KRP's interact withthe microtubules of the mitotic spindle or with the chromosomes directlyand appear to play a pivotal role during the mitotic stages of the cellcycle. These mitotic KRP's are of particular interest for thedevelopment of cancer therapeutics.

Kinesin spindle protein (KSP) (also known as Eg5, HsEg5, KNSL1, orKIF11) is one of several kinesin-like motor proteins that are localizedto the mitotic spindle and known to be required for formation and/orfunction of the bipolar mitotic spindle.

In 1995, the depletion of KSP using an antibody directed against theC-terminus of KSP was shown to arrest HeLa cells in mitosis withmonoastral microtubule arrays (Slangy et al., Cell 83:1159-1169, 1995).Mutations in bimC and cut7 genes, which are considered to be homologuesof KSP, cause failure in centrosome separation in Aspergillus nidulans(Enos. A. P., and N. R. Morris, Cell 60:1019-1027, 1990) andSchizosaccharomyces pombe (Hagan, I. and M. Yanagida, Nature347:563-566, 1990). Treatment of cells with either ATRA (alltrans-retinoic acid), which reduces KSP expression on the protein level,or depletion of KSP using antisense oligonucleotides revealed asignificant growth inhibition in DAN-G pancreatic carcinoma cellsindicating that KSP might be involved in the antiproliferative action ofall trans-retinoic acid (Kaiser, A., et al., J. Biol. Chem. 274,18925-18931, 1999). Interestingly, the Xenopus laevis Aurora-relatedprotein kinase pEg2 was shown to associate and phosphorylate XlEg5(Giet, R. et al., J. Biol. 274:15005-15013, 1999). Potential substratesof Aurora-related kinases are of particular interest for cancer drugdevelopment. For example, Aurora 1 and 2 kinases are over expressed onthe protein and RNA level and the genes are amplified in colon cancerpatients.

The first cell permeable small molecule inhibitor for KSP, “monoastral,”was shown to arrest cells with monopolar spindles without affectingmicrotubule polymerization as do conventional chemotherapeutics such astaxanes and vinca alkaloids (Mayer, T. U, et al., Science 286:971-974,1999), Monastrol was identified as an inhibitor in phenotype-basedscreens and it was suggested that this compound may serve as a lead forthe development of anticancer drugs. The inhibition was determined notto be competitive with respect to adenosine triphosphate interactionwith KSP, and was found to be rapidly reversible (DeBonis. S., et al.,Biochemistry 42:338-349, 2003; Kapoor, T. M., et al., J. Cell Biol.150:975-988, 2000).

In light of the importance of improved chemotherapeutics, there is aneed for KSP inhibitors that are effective in vivo inhibitors of KSP andKSP-related proteins. Some inhibitors of KSP have been reportedpreviously. For example, WO 2006/002236 and PCT/US2006/031129 disclosecertain classes of compounds indicated to be inhibitors of KSP.

A specific kinesin inhibitor compound isN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidewhich has the following chemical structure:

However, after a specific compound is identified as a promisingcandidate for use in a pharmaceutical composition, it is still necessaryto fine-tune its properties with respect to a number of criticalparameters, such as stability in solid state and/or liquid formulations,hygroscopicity, crystallinity, toxicological considerations, meltingpoint, or solubility in water and aqueous media.

Relevant properties of a pharmaceutical compound are affected by thetype of salt and/or crystalline modification. The criteria for theselection of the salt or the crystalline modification depend inter aliaon the planned route(s) of administration.

It has now been surprisingly found that under certain conditions newsalts ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethyl-propyl)-2-hydroxyacetamidecan be provided which have advantageous utilities and properties.

Additionally, it has been surprisingly found that under certainconditions new solid forms ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidecan be provided which are described hereinafter as crystalline forms Dand H and amorphous form A, and which have advantageous utilities andproperties.

Thus, according to a first aspect the present invention provides a saltofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamide,wherein the anion is selected from the group consisting of mesylate,tosylate, hippurate, glycolate, and sulfate.

In a preferred embodiment, the anion is mesylate and the salt is presentas a hydrate.

In the context of the present invention, the term “hydrate” is to beinterpreted according to its commonly accepted meaning. It refers towater molecules incorporated into the crystal structure of a hostcompound and encompasses stoichiometric as well as non-stoichiometrichydrates.

Preferably, the hydrate of the mesylate salt contains water in an amountof 1 to 6 wt % determined by thermogravimetric analysis.

Preferably, the hydrate of the mesylate salt is a hemihydrate(N-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidemesylate×0.5 H₂O), a monohydrate(N-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidemesylate×1 H₂O), sesquihydrate(N-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidemesylate×1.5 H₂O) or a dihydrate(N-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidemesylate×2 H₂O).

According to a further aspect, the present invention provides a processfor the preparation of the salts as defined above, wherein the salt isprecipitated from a polar solvent system.

The salts ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamideas described above can be prepared by precipitation methods known to theskilled person.

The starting compoundN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidefrom which the salts are prepared can be obtained by methods known inthe art and further described below. For example, methods for making thecompound are described in PCT/US2005/022062 (WO 2006/002236) and thecorresponding U.S. patent applications. Examples of additional synthesismethods applicable to the preparation ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamideare provided in the reaction schemes below.

Compound 1.1 and 1.2 were reacted with K₂CO₃ in acetone containing Kl.The use of K₂CO₃/acetone was found to be superior to Cs₂CO₃/ethanolbecause of the lower cost of K₂CO₃ and because compound 1.3 precipitatedfrom the acetone solution upon addition of water, removing the need foran aqueous workup to extract 1.3. Keto ester 1.3 was then refluxed withammonium acetate (NH₄OAc) in toluene to give imidazole 1.4. The use oftoluene was found to afford higher yields of the imidazole in comparisonto refluxing in xylenes with a Dean Stark trap, as the latter method ledto the removal of ammonium acetate from the reaction mixture into thetrap. Reaction of 1.4 with benzylbromide and K₂CO₃ in dimethylformamideafforded 1.5, which can be precipitated from the reaction solution uponaddition of water. Treatment of 1.5 with methanol and acetyl chloridegave the HCl salt of 1.6 which was then converted to its free base whentitrated with a NaOH/methanol solution. The formation of 1.6 from 1.1and 1.2 was found to proceed with 81% yield with high purity (>97% asdetermined by HPLC) and high optical purity (>99% e.e.).

Scheme 2 illustrates the preparation of an aldehyde that can be used inthe reductive amination step to prepareN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamide,

After the reductive amination, known acylating agents and conditions areused to acylate the secondary amine to provide the starting compoundN-((S-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamide,Scheme 3 illustrates the reductive amination. Acylation of the aminefollowed by deprotection of the phthalimide and removal of a protectinggroup on the free hydroxyl group providesN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamide.Suitable protective groups for the hydroxyl include, for example, benzylethers that can be removed by hydrogenolysis and alkyl carbonates thatcan be selectively removed with reagents such as trimethylsilyl iodide.

As indicated above, the salts ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidedescribed above can be prepared by precipitation methods which are inprinciple known to the skilled person.

Preferably, the polar solvent system comprises a polar solvent selectedfrom an alcohol, preferably isopropanol and/or tert-butanol, a nitrile,preferably acetonitrile, acetone, or any mixture thereof. The polarsolvent system may further comprise water.

For intravenous (i.v.) applications, the salt as described above needsto be provided in liquid formulations of sufficient stability.

Thus, according to a further aspect, the present invention provides aliquid formulation, prepared by dissolving or suspending the salt asdefined above in a solvent or suspension medium. Preferably, the saltemployed is the hydrate form D or hydrate form H as further definedbelow.

Preferably, the solvent or suspension medium is water having a pH ofless than 7, more preferably a pH of 6 or less, even more preferably apH of 5.5 or less, optionally further comprising an additionalpharmaceutically acceptable organic solvent dissolving in water.

As indicated above, relevant properties of a pharmaceutical compoundsuch as stability in solid state, hygroscopicity, processability,solubility etc. can be affected by the type of crystalline modification.

According to a further aspect, the present invention provides acrystalline hydrate form D ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamide.In a preferred embodiment, the form D shows the X-ray diffractiondiagram indicated in FIG. 1.

According to a further aspect, the present invention provides acrystalline hydrate form H ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamide.In a preferred embodiment, the form H shows the X-ray diffractiondiagram indicated in FIG. 2.

Preferably, the crystalline form H is prepared by crystallization inacetonitrile, preferably dried acetonitrile, and subsequent isolation inhumid air, for instance as described in the Examples.

Preferably, the crystalline form D is prepared in an acetonitrile/watersolvent system, for instance as described in the Examples.

According to a further aspect, the present invention provides apharmaceutical composition comprising an active component which isselected from the salt as defined above.

Preferably, the pharmaceutical composition is used for the treatment ofa proliferative disease such as cancer.

More preferably, the proliferative disease is selected from a solidtumor or a hematological cancer in a mammal

Preferably, the solid tumor solid tumor is selected from the groupconsisting of lung carcinoma, breast carcinoma, ovarian carcinoma, skincarcinoma, colon carcinoma, urinary bladder carcinoma, liver carcinoma,gastric carcinoma, prostate cancer, renal cell carcinoma, nasopharyngealcarcinoma, squamous cell carcinoma, thyroid papillary carcinoma,cervical carcinoma, small cell lung carcinoma (SCLC), non-small celllung carcinoma, pancreatic cancer, head and neck squamous cell cancerand sarcomas.

The present invention is now described in further detail by the examplesprovided below.

EXAMPLES

X-ray diffractograms of crystalline forms were measured with aPANalytical X'Pert powder diffractometer (Copper Kalpha radiation).

Example 1 Tosylate Salt

The tosylate salt ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidewas prepared in acetonitrile as solvent.

The tosylate salt has a melting temperature T (measured by DSC asmelting onset) of 207° C.

The weight loss (%) on drying, measured by thermogravimetry, of the saltas prepared was 1.12%. If kept for 1 day at 92% relative humidity, theweight loss was 1.71%. This clearly indicates that the tosylate salt isof low hygroscopicity.

Example 2 Mesylate Salt Amorphous Form

The mesylate salt ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamideis prepared in acetonitrile as solvent.

The weight loss (%) on drying, measured by thermogravimetry, of the saltas prepared was 2.08%. If kept for 1 day at 92% relative humidity, theweight loss was 2.21%. This clearly indicates that the mesylate salt isof low hygroscopicity.

Example 3 Hippurate Salt

The hippurate salt ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidewas prepared in an acetone/water solvent system. The hippurate salt hasa melting temperature Tm (measured by DSC as melting onset) of 80° C.

The weight loss (%) on drying, measured by thermogravimetry, of the saltas prepared was 1.76%. If kept for 1 day at 92% relative humidity, theweight loss was 2.14%. This clearly indicates that the hippurate salt isof low hygroscopicity.

Example 4 Sulfate Salt

The sulfate salt ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benztyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidewas prepared in isopropanol as solvent.

The sulfate salt has a melting temperature Tm (measured by DSC asmelting onset) of 98° C.

Example 5 Glycolate Salt

The glycolate salt ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidewas prepared in isopropanol as solvent.

Example 6 Stability of the Salts in the Solid State

Tests for solid state stability were made for the mesylate, tosylate andhippurate salts as prepared above. The results are shown below in Table1.

TABLE 1 Solid state stability Salt Form Tosylate Mesylate HippurateAmount of Amount of Amount of degra- degra- degra- Test dation dationdation Con- products products products ditions [%] Colour [%] Colour [%]Colour 2 weeks, 50° C., tight container Bulk 0.15 A 0.07 A 0.79 A (HPLC)2 weeks, 50° C., 75% r.h. Bulk 0.19 A <0.05 A 1.02 A (HPLC) Xenon light(approx. 1200 kLuxh) Bulk 0.05 A <0.05 A 0.43 A (HPLC) Colour: A: Nochange of colour; B: Slight discolouration; C: Medium discolouration; D:Strong discolouration

The results of Table 1 clearly indicate that the salts are the in thesolid state.

Example 7 Preparation of Liquid Formulations by Dissolving/Suspendingthe Salts

For intravenous (i.v.) applications, stability of the salts in liquidformulations is relevant as well.

From the mesylate, tosylate and hippurate salts as prepared above,liquid formulations were prepared by dissolving/suspending these saltsin a solvent/suspension medium. Stability tests were made on theseliquid formulations. The amount of degradation products and the degreeof discoloration were established. The results are shown below in Table2.

TABLE 2 Stability of liquid formulations Salt Form Tosylate MesylateHippurate Amount of Amount of Amount of degra- degra- degra- Test dationdation dation Con- products products products ditions [%] Colour [%]Colour [%] Colour 0.1% Solutions or suspensions, 1 week, 50° C. pH 30.27 A 0.25 A 0.48 A pH 4.5 0.59 A 0.69 A 1.03 A pH 5 1.81 A 0.71 A 1.80A pH 7 7.60 A 3.87 A 5.32 A Metha- 0.35 A 0.21 A 11.81 A nol 2%Solutions or suspensions, 1 day, room temperature DMSO 0.14 A 0.07 A0.78 A Di- 0.33 A 0.19 A 0.82 A lution, pH 6.8⁽¹⁾ 5% Solutions, 1 day,room temperature DMSO 0.15 A <0.05 A 0.94 A Di- 0.11 A 0.30 A 0.84 Alution, pH 6.8 ⁽¹⁾A portion of the freshly prepared solution/suspensionwas diluted with a buffer pH 6.8 in the ratio 1:100. Colour: A. Nochange of colour; B: Slight discolouration; C: Medium discolouration; D:Strong discolouration

If the pH of the solvent system is below 7, stability can be kept on asufficiently high level.

In methanol, the mesylate and tosylate salts are very stable. In DMSO,either diluted or not, mesylate, tosylate and hippurate salts are verystable.

Example 8 Preparation of crystalline form D ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidemesylate

2.40 g of amorphousN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluoro-phenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidewere dissolved at 50° C. in 9 ml acetonitrile/water 95:5 v/v having awater activity of 0.6, and stirred. Within about 1 minute, a suspensionwas formed. Another 6 ml of acetonitrile/water 95:5 v/v were added.Subsequently, the suspension was cooled to room temperature in about 1hour and stirred for 14 hours. Then, the suspension was filtered and airdried for about 5 minutes. Yield: 1.82 g (75%). The obtained materialwas analyzed by X-ray diffraction (FIG. 1). The solubility of theobtained material in water is 4.4 mg/ml, in buffer with pH 6.8 it is0.010 mg/ml and in 0.1 N HCl it is 1.4 mg/ml.

Example 9 Preparation of crystalline form H ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidemesylate

123 mg of amorphousN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluoro-phenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidewere dissolved in 0.5 ml methanol/water (1:1, v:v) and stirred for oneday at room temperature. The obtained suspension was filtered and driedat the air for approximately 2 minutes. The obtained material wasanalyzed by X-ray diffraction (FIG. 2).

1. A salt ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamide,wherein the anion is selected from the group consisting of mesylate,tosylate, hippurate, glycolate, and sulfate.
 2. The salt according toclaim 1, wherein the anion is mesylate and the salt is a hydrate.
 3. Thesalt according to claim 2, wherein the hydrate is a hemihydrate(N-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidemesylate×0.5 H₂O), a monohydrate(N-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidemesylate×1 H₂O), sesquihydrate(N-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidemesylate×1.5 H₂O) or a dihydrate(N-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidemesylate×2 H₂O).
 4. A crystalline hydrate form D ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidemesylate.
 5. A crystalline hydrate form H ofN-((S)-3-amino-4-fluorobutyl)-N-((R)-1-(1-benzlyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyacetamidemesylate.
 6. A process for the preparation of one of the crystallineform H according to claim 5, wherein such form is prepared bycrystallization in acetonitrile and subsequent isolation in humid air.7. A liquid formulation, prepared by dissolving or suspending at leastone salt according to claim 1 in a solvent or suspension medium, whichcomprises water having a pH of less than 7.